| Living organisms have evolved a myriad of effective antioxidant systems to adapt to an oxygen-rich environment and protect themselves against the toxicity of reactive oxygen species (ROS). Heme oxygenase (HO) is an enzyme that has been found to be critical for protection from the oxidative stress of a wide variety of conditions and disease states, including atherosclerosis, stroke, transplant rejection, and hyperoxic lung injury. Though the anti-inflammatory, cytoprotective, and anti-apoptotic effects of HO are well-known, the mechanism by which HO confers its protection is unclear. This thesis examines this mechanism by studying the physiologic roles of HO's products, iron and bilirubin. Previous analysis of the HO1 −/− mice reveals that HO1 regulates iron homeostasis. While molecular mechanisms for iron entry and storage within cells have been elucidated, no system to mediate iron efflux has been heretofore identified. We now describe an ATP-requiring iron transporter in mammalian cells. The Fe-ATPase is localized together with heme oxygenase-1 to microsomal membranes with both proteins greatly enriched in the spleen. Iron treatment markedly induces Fe-ATPase activity in RAW 264.7 macrophage cells with an initial phase that is resistant to cycloheximide and actinomycin D and a later phase that is inhibited by these agents. Iron release, elicited in intact rats by glycerol-induced rhabdomyolysis, induces Fe-ATPase activity in the kidney. HO1 −/− mice display augmented Fe-ATPase activity in those tissues that accumulate iron. Bilirubin, another product of heme catabolism, is insoluble and toxic with no known physiologic function. Bilirubin possesses antioxidant capabilities, and has been implicated in neuroprotection, as cytotoxicity is exacerbated in brain cultures from HO2 −/− mice. We now establish a major physiologic cytoprotectant, antioxidant role for bilirubin. Depletion of bilirubin, achieved by deletion of its biosynthetic enzyme, biliverdin reductase, using mammalian RNA interference, causes a substantial increase in ROS and augments cell death. Depletion of glutathione, previously accepted as the principal cellular antioxidant cytoprotectant, causes lesser effects. In intact cells low nM concentrations of bilirubin counteract the oxidant effects of 10,000 times higher levels of hydrogen peroxide, because bilirubin acts catalytically, with biliverdin reductase regenerating bilirubin. |
